Effects of cholesterol-lowering probiotic fermentation on the active components and in vitro hypolipidemic activity of sea buckthorn juice.

Sea buckthorn has lipid-lowering properties and is widely used in the development of functional foods. In this study, a probiotic (Lactobacillus plantarum, Lp10211) with cholesterol-lowering potential and acid and bile salt resistant was screened for the fermentation of sea buckthorn juice. Changes in the active ingredients, such as sugars and phenolics, before and after fermentation, as well as their in vitro lipid-lowering activities, were compared. The contents of reducing and total sugars decreased substantially after fermentation. Lp10211 primarily utilized fructose for growth and reproduction, with a utilization rate of 76.9%. The phenolic compound content of sea buckthorn juice increased by 37.06% after fermentation and protected the phenolic components from degradation (protocatechuic and p-coumaric acids) and produced new polyphenol (shikimic acid). Enhanced inhibition of pancreatic lipase activity (95.42%) and cholesterol micellar solubility (59.15%) was evident. The antioxidant properties of the fermentation broth were improved. Notably, Lp10211 preserved the color and reversed browning in sea buckthorn juice. The collective findings indicate that fermentation of sea buckthorn juice by Lp10211 may enhance the functional components and lipid-lowering activity of sea buckthorn, which may provide a new approach for the development of lipid-lowering foods.

Isolation and characterization of feruloylated oligosaccharides from Phyllostachys acuta and in vitro antioxidant activity.

Feruloylated oligosaccharides (FOs) generated by decomposing plant hemicellulose, offer a wide range of potential applications in both the food and biomedical areas. As a graminaceous plant, bamboo is rich in hemicellulose. However, the structural composition and activity studies of FOs from it were rarely reported. In this study, FOs from Phyllostachys acuta (pFOs) obtained by enzymatic hydrolysis were isolated by AmberliteXAD-2 and C18 SPE columns. Then, pFOs were qualitatively and quantitatively analyzed by UPLC-ESI-MS/MS after labeled by 3-Amino-9-ethyl-carbazole (AEC), and the chemical antioxidant activity of pFOs and effects of pFOs on H2O2-induced oxidative damage were investigated. Finally, 14 of pFOs isomers were distinguished and identified, of which 10 did not contain hexoses and 4 did, and the three most abundant pFO structures were 12 (Iso 7, F1A1X2H2-AEC, 29.04 %), 11 (Iso 6, F1A1X1H2-AEC, 17.96 %), and 4 (Iso 3-1, F1A1X3-AEC, 15.57 %). The results of antioxidant studies showed that pFOs possessed certain reducing power, scavenging DPPH radicals, scavenging superoxide anion radicals, and scavenging hydroxyl radicals. Among them, the ability to clear DPPH radicals was particularly significant. pFOs significantly reduced the viability of RAW264.7 cells after H2O2 induction, whereas pFOs had a significant protective effect (p < 0.001). pFOs increased the viability of T-AOC and SOD enzymes in oxidatively damaged cells, as well as had a significant inhibition effect on ROS elevation (p < 0.001). This study lays the foundation for the structural analysis and antioxidant activity evaluation of bamboo-derived feruloyl oligosaccharides for their application in food and pharmaceutical fields.

Biosynthesis of Feruloyl Glycerol from Ferulic Acid and Glycerol Through a Two-Enzyme Cascade Reaction.

Feruloyl glycerol (FG) has a variety of biological activities, but the green synthesis methods of FG remain rare. In this study, FG was prepared by a cascade reaction catalyzed by 4-coumarate coenzyme A ligase (4CL) and hydroxycinnamoyl acyltransferase 4 (HCT4). The cascade reaction carried out at solvent water and room temperature is more convenient and greener. Firstly, the product derived from the cascade reaction was characterized by TLC, HPLC, FTIR, and ESI-MS. The results showed that the product was FG. Secondly, the effects of temperature, pH, enzyme ratio, Mg2+ concentration, and CoA concentration on the cascade reaction were investigated. Consequently, the highest reaction rate was obtained at 30 °C, pH 6, an enzyme ratio of 1:3, and Mg2+ concentration of 5 mM. Finally, semi-preparative scale synthesis for FG was conducted. The production of FG reached 35.1 mM at 24 h with the FG conversion of 70.18%. In a word, a novel idea for the efficient and green synthesis of FG was proposed, which had great potential for industrial application.

Structural characterization and potential anti-tumor activity of a polysaccharide from the halophyte Salicornia bigelovii Torr.

Plant polysaccharides are highly potent bioactive molecules. Clarifying the structural composition and bioactivities of plant polysaccharides will provide insights into their structure-activity relationships. Therefore, herein, we identified a polysaccharide produced by Salicornia bigelovii Torr. and analyzed the structure and anti-tumor activity of its component, SabPS-1. SabPS-1 was 3.24 × 104 Da, primarily composed of arabinose (24.96 %), galactose (30.39 %), and galacturonic acid (23.20 %), rhamnose (6.21 %), xylose (4.99 %), glucuronic acid (3.12 %), mannuronic acid (1.75 %), mannose (1.69 %), glucose (1.54 %), fucose (1.12 %), and guluronic acid (1.03 %). The backbone of SabPS-1 was a â†’ 4)-ß-D-GalpA-(1→, →5)-α-L-Araf-(1→, and→4)-ß-D-Galp-(1 â†’ molecule with a branched chain of α-L-Araf-(1 â†’ connected to sugar residues of →3,6)-ß-D-Galp-(1 â†’ in the O-3 position. SabPS-1 induced apoptosis and inhibited the growth of HepG-2 cells, with viability of 47.90 ± 4.14 (400 µg/mL), indicating anti-tumor activity. Apoptosis induced by SabPS-1 may be associated with the differential regulation of caspase 3, caspase 8, Bax, and Bcl-2. To the best of our knowledge, this is the first study to investigate the principal structures and anti-tumor biological activities of SabPS-1. Our findings demonstrated the excellent anti-tumor properties of SabPS-1, which will aid in the development of anti-tumor drugs utilizing Salicornia bigelovii Torr.

Optimization of Microwave-Assisted Extraction Process of Total Flavonoids from Salicornia bigelovii Torr. and Its Hepatoprotective Effect on Alcoholic Liver Injury Mice.

The objective of this study was to determine the optimal extraction conditions for total flavonoids from S. bigelovii using microwave-assisted extraction and to analyze the protective effect of total flavonoids from S. bigelovii on alcoholic liver injury in mice. The optimization of the process conditions for the microwave-assisted extraction of total flavonoids from S. bigelovii was performed using response surface methodology, and an alcohol-induced acute liver injury model in mice was used to investigate the effects of different doses of total flavonoids (100 mg/kg, 200 mg/kg, and 400 mg/kg) on the levels and activities of serum alanine aminotransferase kits (ALT), glutamic oxaloacetic transaminase kits (AST), superoxide dismutase kits (SOD), glutathione peroxidase kits (GSH-Px), and malondialdehyde (MDA). We performed hematoxylin-eosin (H&E) staining analysis on pathological sections of mouse liver tissue, and qRT-PCR technology was used to detect the expression levels of the inflammatory factors IL-1 ß, IL-6, and TNF-α. The results revealed that the optimal extraction process conditions for total flavonoids in S. bigelovii were a material-to-liquid ratio of 1:30 (g/mL), an ethanol concentration of 60%, an extraction temperature of 50 °C, an ultrasound power of 250 W, and a yield of 5.71 ± 0.28 mg/g. Previous studies have demonstrated that the flavonoids of S. bigelovii can significantly inhibit the levels of ALT and AST in the serum (p < 0.001), reduce MDA levels (p < 0.001), increase the activity of the antioxidant enzymes SOD and GSH-Px (p < 0.001), and inhibit the IL-1 ß, IL-6, and TNF-α gene expression levels (p < 0.001) of inflammatory factors. The total flavonoids of S. bigelovii exert a protective effect against alcoholic liver injury by reducing the levels of inflammation, oxidative stress, and lipid peroxidation caused by alcohol. The results of this study lay the foundation for the high-value utilization of S. bigelovii and provide new resources for the development of liver-protective drugs.

A green method for decolorization of polysaccharides from alfalfa by S-8 macroporous resin and their characterization and antioxidant activity.

In this study, the decolorization conditions of polysaccharides extracted from alfalfa by S-8 macroporous adsorption resin were optimized through the response surface method, and the physicochemical properties and antioxidant activity of decolorized polysaccharides were investigated. The optimal decolorization conditions were determined to be as follows: the amount of S-8 macroporous adsorption resin was 1.4 g, the adsorption time was 2 h, and the adsorption temperature was 58 °C. Under these optimal conditions, a decolorization ratio of 71.43 ± 0.23% was achieved, which was consistent with the model hypothesis. The adsorption curve showed that S-8 macroporous adsorption resin adsorption of pigment molecules in alfalfa polysaccharides (APS) agreed with the Freundlich and pseudo-second-order equations, and the adsorption was a spontaneous endothermic process. High-performance liquid chromatography (HPLC) analysis of monosaccharide composition showed that APS was composed of mannose, glucose, galactose, arabinose and glucuronic acid in a molar ratio of 1.18 : 8.04 : 1.22 : 0.92 : 1. The results of antioxidant activity studies showed that APS had strong scavenging activity against ABTS, DPPH and hydroxyl radicals. This study will help to further understand the adsorption mechanism of macroporous resin on polysaccharide pigment molecules and lay a basis for evaluating their physiological activity.

Molecular cloning, expression, and functional analysis of copper amine oxidase gene from mulberry (Morus alba L.).

In this study, we investigated a key enzyme encoded by the gene copper amine oxidase (MaCAO), which is involved in the biosynthetic pathway of 1-deoxynojirimycin (DNJ)1, an active ingredient in mulberry leaves. The 1680 bp long MaCAO was successfully cloned (GenBank accession no: MH205733). Subsequently, MaCAO was heterologously expressed using a recombinant plasmid, pET-22b (+)/MaCAO in Escherichia coli BL21 (DE3). A protein with a molecular mass of 62.9 kDa was obtained, whose function was validated through enzymatic reaction. Bioinformatics analysis identified that MaCAO contained the same conserved domain as that of copper amine oxidases ("NYDY"). Furthermore, the tertiary structure of the predicted protein using homology modeling revealed 46% similarity with that of copper amine oxidase (Protein Data Bank ID: 1W2Z). Gas chromatography-mass spectrometry analysis of the enzymatic reaction revealed that MaCAO could catalyze 1,5-pentanediamine to produce 5-aminopentanal. Additionally, levels of mulberry leaf DNJ content were significantly positively correlated with expression levels of MaCAO (P < 0.001). Our results conclude that MaCAO is the key enzyme involved in the biosynthetic pathway of DNJ. The function of MaCAO is validated, providing a foundation for the further analysis of biosynthetic pathways of DNJ in mulberry leaves using tools of synthetic biology.

Effects of rice bran feruloyl oligosaccharides on gel properties and microstructure of grass carp surimi.

Effects of feruloyl oligosaccharides (FOs) from rice bran on the gel properties, microstructure, and sensory properties of grass crap surimi gel were investigated. The results showed that FOs decreased the whiteness of surimi gel, and improved the water-holding capacity and breaking force of surimi gel. According to the texture analysis, the hardness and chewiness of surimi gel significantly increased by adding 0.3% FOs, but had no significant effect on the springiness and cohesiveness. The changes in AFM images indicated that FOs made myofibrillar protein aggregated and uniformly distributed. The SEM micrograph revealed that the 0.3% FOs group had the most compact and ordered network structure. Additionally, sensory characteristics suggested that FOs reduced off-odor from freshwater fish and remained fish delicious taste. This study provides a new prospect for the potential commercial application of FOs as a health gel enhancer in surimi products.

Antioxidant properties of water-soluble polysaccharides prepared by co-culture fermentation of straw and shrimp shell.

Herein, we present a method for producing water-soluble polysaccharides (WSPs) by co-culture fermentation of straw and shrimp shells. The chitin-degrading strain was isolated and genotypically identified as the non-pathogen Photobacterium sp. LYM-1 in this study. Photobacterium sp. LYM-1 and Aureobasidium pullulans 2012 could coexist without antagonism. WSPs concentrations were higher in co-culture fermentations of Photobacterium sp. LYM-1 and A. pullulans 2012 (PsL/AP-WSPs) compared to monocultures (PsL-WSPs and AP-WSPs). FTIR was used to examine the polysaccharide properties of three WSP fractions. The monosaccharide compositions of three WSPs fractions were primarily composed of mannose, ribose, glucosamine, glucose, galactose, and arabinose with varying molecular weights and molar ratios according to HPLC analysis. PsL/AP-WSPs showed better scavenging effects on DPPH, ABTS, and OH free radicals, demonstrating the application potential of PsL/AP-WSPs from straw and shrimp shells. The maximum yield obtained under optimum conditions (fermentation time of 6 days, temperature of 31°C, inoculum concentration of 10% (w/v), and inoculum composition of 2:1) was 5.88 ± 0.40 mg/mL, based on the PsL/AP-WSPs production optimization by orthogonal design. The results suggest that an environmentally friendly approach for WSPs production from agro-food wastes straw and shrimp shells was developed.

Buffer Coefficient as a Predictor of the Prognosis of Massive Cerebral Infarction.

OBJECTIVE: To evaluate the prognostic value of the buffer coefficient, calculated as the ratio of the buffer volume (volume of intracranial cerebrospinal fluid) at the peak of brain edema to the baseline brain volume, and some other parameters in patients with massive cerebral infarction (MCI). METHODS: The cohort comprised 161 patients with MCI who were divided into good and poor prognosis groups according to modified Rankin Scale score at 90 days after onset. Differences in clinical and imaging parameters between these groups were analyzed by univariate analysis, and multifactorial binary logistic regression analysis was used to further identify influencing factors that were significantly different. Receiver operating characteristic curve was used to evaluate the diagnostic performance between the buffer volume and the buffer coefficient. RESULTS: The findings showed that a history of atrial fibrillation, intravenous tissue-type plasminogen activator administration, successful reperfusion, successful craniectomy, low-density lesion volume, brain volume, buffer volume, and buffer coefficient were significantly different between the poor and good prognosis groups (P < 0.05 for all comparisons). Multifactorial binary logistic regression analyses revealed that patients who had large low-density lesion volume and patients who had not achieved successful reperfusion or received intravenous tissue-type plasminogen activator were likely to have a poor prognosis (P < 0.05). The buffer coefficient was identified as an independent predictive factor for MCI (P < 0.001). The area under the receiver operating characteristic curve for the buffer coefficient was 0.862. When the cutoff value was 9.3%, sensitivity of predicting poor prognosis of patients with MCI was 94.7%. CONCLUSIONS: The buffer coefficient has potential benefits as a prognostic indicator for MCI that can be used to detect even subtle changes in brain edema.

Large Relative Surface Area of Hematomas Predict a Poor Outcome in Patients with Spontaneous Intracerebral Hemorrhage.

OBJECTIVE: The aim of this study is to investigate the predictive value of relative surface area of hematoma on poor prognosis of spontaneous intracerebral hemorrhage (sICH). MATERIALS AND METHODS: We retrospectively analyzed 102 patients with sICH who met the inclusion criteria, attending to Yongchuan Hospital of Chongqing Medical University from January 2019 to September 2020. Cranial CT within 6 h of onset was completed and repeated in 24 h. The volume and surface area of the hematoma are measured by software, and the relative surface area is calculated from the above data. The primary outcome was 90-day modified Rankin Scale(mRS) score, which was classified as good prognosis (≤2 points) and poor prognosis (>2 points) according to the results. Univariate analysis and binary logistic regression analysis were used to calculate the effect of each variable on poor prognosis. RESULTS: The study is comprised of 52 patients with favorable functional prognosis and 50 patients with unfavorable one. The findings showed that admission National Institutes of Health Stroke Scale(NIHSS), hematoma volume, relative surface area, hematoma expansion(HE) and serum calcium concentration were independent influencing factors for poor prognosis(P=0.004,0.007,0.023,0.001,0.035, respectively). ROC curve analysis revealed that the area under the curve of the relative surface area of the hematoma predicted poor prognosis was 0.894(95% CI 0.830-0.985, P<0.001), which was better than the rest of the variables. CONCLUSIONS: The relative surface area of hematoma in sICH is an independent risk factor for poor prognosis with a high predictive value, and large relative surface area suggests poor prognosis.

Heterologous Expression of a Thermostable Chitinase from Myxococcus xanthus and Its Application for High Yield Production of Glucosamine from Shrimp Shell.

Glucosamine (GlcN) is a widely used food supplement. Hence, enormous attention has been concerned with enzymatic production of GlcN owing to its advantage over a chemical approach. In this study, a previously unstudied chitinase gene (MxChi) in the genome of Myxococcus xanthus was cloned, expressed in recombinant soluble form and purified to homogeneity. TLC-, UPLC-, and microplate-reader- based activity tests confirmed MxChi hydrolyzes colloidal chitin to chitobiose as sole product. The optimal catalytic pH and temperature of MxChi was identified as 7.0 and 55 °C, respectively. MxChi exhibited 80% activity after 72 h incubation at 37 °C. The site-directed mutagenesis revealed that the amino acids D323A, D325A, and E327A of MxChi were in the DXDXE catalytic motif of GH18. When coupled with ß-N-acetylhexosaminidase (SnHex) and deacetylase (CmCBDA), the enzyme allowed one-pot extraction of GlcN from colloidal chitin and shrimp shell. The optimal condition was 37 °C, pH 8.0, and 1/3/16.5 (MxChi/SnHex/CmCBDA), conducted by orthogonal design for the enzymatic cascades. Under this condition, the yield of GlcN was 26.33 mg from 400 mg shrimp shell. Facile recombinant in E. coli, robust thermostability and pure product herein makes newly discovered chitinase a valuable candidate for the green recycling of chitin rich waste.

In Vivo Functional Verification of Four Related Genes Involved in the 1-Deoxynojirimycin Biosynthetic Pathway in Mulberry Leaves.

The alkaloid 1-deoxynojirimycin (DNJ) is one of the major bioactive compounds in mulberry leaves (Morus alba L.). Previously, we discovered four key genes involved in the pathway from lysine to piperidine in the biosynthesis of DNJ in mulberry leaves, MaLDC (MG727866), MaCAO (MH205733), MaSDR1 (MT989445), and MaSDR2 (MT989446), which encoded lysine decarboxylase, copper amine oxidase, and short-chain dehydrogenase/reductase 1 and 2, respectively. However, the in vivo functions of these four genes have not been verified yet. Here, these four genes were successfully cloned and used for the establishment of C58C1 Agrobacterium rhizogenes mediated overexpression genetic transformation systems and GV3101 Agrobacterium-mediated virus-induced gene silencing transformation systems in order to verify the influence of these four genes on the biosynthetic content of DNJ in mulberry leaves. The results showed that the content of DNJ increased after the four genes were overexpressed. When these four genes were silenced, the gene expression was blocked, which affected the biosynthesis of DNJ, and the DNJ content decreased. The above results indicated that these four genes participated in DNJ biosynthesis. This study provided a foundation for further elucidating the regulatory mechanisms of DNJ biosynthesis in mulberry leaves.

Comparative Transcriptome Analysis of Key Reductase Genes Involved in the 1-Deoxynojirimycin Biosynthetic Pathway in Mulberry Leaves and Cloning, Prokaryotic Expression, and Functional Analysis of MaSDR1 and MaSDR2.

The alkaloid 1-deoxynojirimycin (DNJ) is the main bioactive ingredient in the hypoglycemic action of mulberry leaves (Morus alba L.). Our previous research clarified the upstream pathway from lysine to Δ1-piperideine in the biosynthesis of DNJ in mulberry leaves, but the pathway and related reductase genes from Δ1-piperideine to piperidine are still unclear. Here, a comparative transcriptome was used to analyze the transcriptome data of two samples (July and November) of mulberry leaves with significant differences in the content of DNJ and screen-related reductase genes. Results showed that expression levels of MaSDR1 and MaSDR2 were significantly and positively correlated with the content of DNJ (P < 0.05) in different seasons. MaSDR1 (GenBank accession no. MT989445) and MaSDR2 (GenBank accession no. MT989446) were successfully cloned and used for prokaryotic expression and functional analysis in vitro. MaSDR1 and MaSDR2 could catalyze the reaction of Δ1-piperideine with the coenzyme NADPH to generate piperidine. The kinetic parameters of MaSDR1 and MaSDR2 indicated that MaSDR2 had a higher binding ability to Δ1-piperideine than MaSDR1. This study provided insights into the biosynthesis of DNJ in mulberry leaves.

Overexpression of OsMYB305 in Rice Enhances the Nitrogen Uptake Under Low-Nitrogen Condition.

Excessive nitrogen fertilizer application causes severe environmental degradation and drives up agricultural production costs. Thus, improving crop nitrogen use efficiency (NUE) is essential for the development of sustainable agriculture. Here, we characterized the roles of the MYB transcription factor OsMYB305 in nitrogen uptake and assimilation in rice. OsMYB305 encoded a transcriptional activator and its expression was induced by N deficiency in rice root. Under low-N condition, OsMYB305 overexpression significantly increased the tiller number, shoot dry weight and total N concentration. In the roots of OsMYB305-OE rice lines, the expression of OsNRT2.1, OsNRT2.2, OsNAR2.1, and OsNiR2 was up-regulated and 15NO3 - influx was significantly increased. In contrast, the expression of lignocellulose biosynthesis-related genes was repressed so that cellulose content decreased, and soluble sugar concentration increased. Certain intermediates in the glycolytic pathway and the tricarboxylic acid cycle were significantly altered and NADH-GOGAT, Pyr-K, and G6PDH were markedly elevated in the roots of OsMYB305-OE rice lines grown under low-N condition. Our results revealed that OsMYB305 overexpression suppressed cellulose biosynthesis under low-nitrogen condition, thereby freeing up carbohydrate for nitrate uptake and assimilation and enhancing rice growth. OsMYB305 is a potential molecular target for increasing NUE in rice.

Antioxidant and anti-aging activities of polysaccharides from Cordyceps cicadae.

Cordyceps cicadae is a traditional Chinese medicine with high nutritional value and biological activities. Previously, we reported on the antioxidant activity associated with the polysaccharides from Cordyceps cicadae (CP). To further explore which of the fraction of CP had the greatest potency, in here, the in vitro antioxidant and in vivo anti-aging activities of the fractions CP30-CP80 of CP were evaluated. The in vitro antioxidant activity results revealed that all the fractions (i.e. CP30-CP80) were potent with CP70 as the most potent. Notably, CP70 prolonged the lifespan of Drosophila (P < 0.05), increased the activities of catalase (CAT) and glutathione peroxidase (GSH-Px) (P < 0.01), and inhibited the formation of malondialdehyde (MDA) (P < 0.01). Additionally, CP70 upregulated the expression level of antioxidant-related genes CAT, SOD1 and MTH in Drosophila (P < 0.05). These results indicated that CP70 may prolong the lifespan of Drosophila through the up-regulation of the expression level of antioxidant-related genes CAT, SOD1 and MTH in Drosophila. Thus, polysaccharides from Cordyceps cicadae possess significant antioxidant and anti-aging activities, and could be explored as a new dietary supplement to slow down the aging process.

Transcriptomic analysis of key genes involved in chlorogenic acid biosynthetic pathway and characterization of MaHCT from Morus alba L.

Mulberry leaves (Morus alba L.) are of high medicinal value in traditional Chinese medicine with chlorogenic acid (CGA) as its major biologically active constituent. Mulberry leaves require that they be harvested after frost; previous studies have shown CGA accumulation significantly increased after frost. However, the molecular mechanism of how frost changes the CGA content in mulberry leaves is unclear. Additionally, the mechanism of CGA biosynthesis and key genes in mulberry leaves are not well-understood. In this study, transcriptome sequencing was performed on two mulberry leaf samples with different CGA contents (before and after frost). Fifty-eight genes were annotated in the CGA biosynthetic pathway. Compared to those in pre-frost mulberry leaves, 12 and 5 genes were upregulated and downregulated, respectively, in post-frost leaves. Correlation analysis showed that the expression levels of four genes were significantly positively correlated with CGA content, including those encoding phenylalanine ammonia-lyase, 4-coumarate-CoA ligase, hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferase (HCT), and coumaroyl quinate/shikimate 3'-hydroxylase, and may be key genes in the CGA biosynthetic pathway. We cloned MaHCT4 (GenBank accession no. MH476577) from mulberry leaves. Multiple sequence alignment suggested that MaHCT4 contains the conserved domains HXXXD and DFGWG. Enzymatic assays indicated that MaHCT4 catalyzes the formation of p-coumaroyl shikimic acid, p-coumaroyl quinic acid, and CGA. The Km values of quinic acid and shikimic acid were 10 ±â€¯1.0 and 31 ±â€¯1.7 µM, respectively, suggesting that MaHCT4 favored quinic acid over shikimic acid as its acyl acceptor. Using quinic acid as an acyl acceptor, MaHCT4 showed a preference for p-coumaroyl-CoA over caffeoyl-CoA. Our results provide insight into the molecular mechanism of how frost alters the CGA content and roles of key genes involved in the CGA biosynthetic pathway in mulberry leaves.

Characterization and anti-tumor activity of a polysaccharide isolated from Dendrobium officinale grown in the Huoshan County.

BACKGROUND: Polysaccharides are carbohydrate chains composed of linked monosaccharide units. Accumulating studies report that polysaccharides isolated from Dendrobium officinale have a variety of functions. However, the composition and anti-tumor activity of D. officinale grown in the Huoshan area are largely unknown. METHODS: A polysaccharide (DOPA-1) was isolated from D. officinale by hot water extraction and ethanol precipitation, followed by purification via DEAE-cellulose and Sephadex G-100 chromatography. DOPA-1 was analyzed by infrared and nuclear magnetic resonance and then characterized by periodate oxidation and Smith degradation. The anti-tumor activity of DOPA-1 was then tested in HepG-2 cells. RESULTS: Our results show that DOPA-1 is mainly comprised of mannose, glucose, and galactose at a molar ratio of 1:0.42:0.27 and has an average molecular weight of 2.29 × 105 Da. Additionally, DOPA-1 inhibited HepG-2 cell growth in a dose-dependent manner. DOPA-1-treated HepG-2 cells also had increased reactive oxygen species (ROS) levels and decreased mitochondrial membrane potential. Furthermore, apoptosis was observed in DOPA-1-treated HepG-2 cells along with Bcl-2 downregulation and Bax upregulation at the protein level. CONCLUSIONS: Our findings suggest that DOPA-1 induces apoptosis in tumor cells via altered mitochondrial function, ROS production, and altered apoptosis-related protein expression. This bioactive polysaccharide could, therefore, potentially be further developed as an anti-tumor adjuvant drug.

Transcriptome analysis and identification of key genes involved in 1-deoxynojirimycin biosynthesis of mulberry (Morus alba L.).

Mulberry (Morus alba L.) represents one of the most commonly utilized plants in traditional medicine and as a nutritional plant used worldwide. The polyhydroxylated alkaloid 1-deoxynojirimycin (DNJ) is the major bioactive compounds of mulberry in treating diabetes. However, the DNJ content in mulberry is very low. Therefore, identification of key genes involved in DNJ alkaloid biosynthesis will provide a basis for the further analysis of its biosynthetic pathway and ultimately for the realization of synthetic biological production. Here, two cDNA libraries of mulberry leaf samples with different DNJ contents were constructed. Approximately 16 Gb raw RNA-Seq data was generated and de novo assembled into 112,481 transcripts, with an average length of 766 bp and an N50 value of 1,392. Subsequently, all unigenes were annotated based on nine public databases; 11,318 transcripts were found to be significantly differentially regulated. A total of 38 unique candidate genes were identified as being involved in DNJ alkaloid biosynthesis in mulberry, and nine unique genes had significantly different expression. Three key transcripts of DNJ biosynthesis were identified and further characterized using RT-PCR; they were assigned to lysine decarboxylase and primary-amine oxidase genes. Five CYP450 transcripts and two methyltransferase transcripts were significantly associated with DNJ content. Overall, the biosynthetic pathway of DNJ alkaloid was preliminarily speculated.

Cloning, expression, and functional analysis of lysine decarboxylase in mulberry (Morus alba L.).

1-Deoxynojirimycin (DNJ) is the main bioactive compound of Morus alba L.. DNJ has pharmacological effects, including blood sugar level regulation and antiviral activity. In this study, the mulberry lysine decarboxylase gene (MaLDC), which is involved in the biosynthesis of DNJ alkaloids, was cloned, expressed, and functionally verified. MaLDC was induced and expressed in Escherichia coli BL21 (DE3). The recombinant soluble MaLDC protein had a relative molecular mass of 24.0 kDa. The protein was purified by Ni-NTA separation. The results showed that MaLDC protein could catalyze lysine decarboxylation to produce cadaverine. The Km and Vmax values were 19.2 µM and 3.31 µM/min, respectively. Quantitative real-time reverse transcription polymerase chain reaction revealed that MaLDC expression was positively correlated with DNJ content (P < 0.001), indicating that the MaLDC could encode a functional protein involved in the biosynthesis of DNJ alkaloid in mulberry. Our results provided a foundation for further studies of the enzymatic properties of LDC and established a basis for the analysis of key enzymes involved in the biosynthetic pathway of mulberry DNJ alkaloid.